Hematopoiesis is a complex hierarchically-organized developmental system where progressive states are governed by properly balanced cell-fate decisions. The outcome of these processes is the continuous and lifelong production of at least eight distinct lineages of mature blood cells. The origin of the hematopoietic hierarchy lies in a rare population of self-renewing multipotent stem cells. Much biological and physical information has been obtained which collectively describes hematopoietic stem cells. In contrast, very little is known about the molecular biology of these cells. There have been few insights into the components and overall nature of the regulatory machinery which orchestrates the correct balance of self-renewal vs. commitment,: as well as other important developmental choices. A molecular understanding of stem cell regulation will provide a new context within which to view a normal or pathological hematopoietic process. In this proposal a range of existing and newly-emerging molecular technologies are focused on the biology of murine hematopoietic stem cells. In Aim One the gene-expression patterns which define the stem cell will be analyzed and will provide a first global stem cell molecular phenotype or "genetic space." To rationally explore the molecular phenotype, powerful bioinforrnatic tools will be applied. A major goal is to identify and characterize all gene-products whose expression segregates to the primitive stem/progenitor cell hierarchy. In Aim Two novel microarray approaches will be employed used to monitor the expression fluctuations in the stem cell "genetic space" as a function of differences in well&#8209;defined biological properties. In all of these studies an emphasis is placed on molecular correlations with quantitatively measured functional activities of the stem cells rather than with their physical properties. Strategies will be developed to measure fluctuations as the stem/progenitor cell hierarchy develops in time. In Aim Three the biological roles of three novel cell-surface molecules isolated from stem cells will be defined using immunochemical and genetic gain and loss- of-function approaches. Collectively, the proposed studies will lay a necessary and firm foundation upon which to unravel stem cell regulation. The conserved properties of the murine and human hematopoietic systems suggest that the proposed studies could have a direct impact on human stem cell transplantation, ex vivo expansion, gene therapy and the etiology of leukemias as well as other disorders.